Inflammation is a biological response to injury, infection or irritation in which a cascade of cellular and microvascular reactions serves to eradicate the infection, remove damaged tissue and generate new tissue. During this process, elevated permeability in microvessels allows neutrophils and mononuclear cells to leave the intravascular compartment, and perform various anti-microbial activities to eradicate the injury. The final stage of inflammation is resolution, a process characterized by active blockade of leukocyte infiltration, followed by their apoptosis and the removal of cellular and molecular debris from inflamed sites, enabling tissue to return to homeostasis.
Many inflammatory diseases fail to resolve, resulting in an excessive and potentially harmful inflammatory response. Such excessive responses are implicated in a number of common diseases such as cancer, asthma, atherosclerosis, autoimmune diseases, Alzheimer's and Parkinson's disease, among others.
A hallmark of the resolution phase is the removal of apoptotic cells, mediated by phagocytes such as macrophages and dendritic cells. In contrast to the clearance of pathogens by phagocytosis which commonly triggers an immune response, the engulfment of apoptotic cells is generally considered anti-inflammatory.
Resolution is considered a biosynthetically active process and a number of key factors and chemical mediators have been found to influence its development, including lipoxins, resolvins, protectins, maresins, eicosanoids, polyunsaturated fatty acids (PUFAs), glucocorticoids, annexin and ω-3 fatty acids, among others. Many of these mediators promote resolution by stimulating macrophage uptake of apoptotic neutrophils and preventing necrosis-driven secondary inflammation.
Some of the molecular features underlying the resolution of acute inflammation have been disclosed by some of the inventors of the present invention (Bannenberg et al. J. Immunol. 2005, 1; 174(7):4345-55) and include specific anti-inflammatory mediators such as lipoxin, resolvins, protectins and intermediates in their generation as molecular pathways of resolution.
A new population of macrophages, typically referred to as CD11blow macrophages, has recently been identified by some of the inventors of the present invention (Schif-Zuck et al. Eur J. Immunol. 2011 41(2):366-79). These macrophages display pro-resolving properties during the resolution of an acute inflammatory response such as increased engulfment of apoptotic leukocytes compared to other classes of macrophages, among other features.
Among the various factors found to take part in inflammatory processes is the 80 kDa glycoprotein lactoferrin. Lactoferrin belongs to the transferrin protein family, characterized by the ability to bind and transfer Fe3+ions. Three different isoforms of lactoferrin have been isolated: lactoferrin-α, capable of iron binding but having no ribonuclease activity, and lactoferrin-β and γ, which possess ribonuclease activity, but are not capable of binding iron. Lactoferrin comprises a single polypeptide chain of about 700 amino acids in length folded into two globular lobes, the C- (carboxy) and N- (amino) terminal regions.
The primary cells involved in lactoferrin synthesis are myeloid cells and secretory epithelia. The highest levels of lactoferrin are found in colostrums, milk and seminal plasma; lactoferrin is also found in most mucosal secretions such as uterine fluid, vaginal secretion, saliva, bile, pancreatic juice, small intestine secretions, nasal secretion, and tears. In addition, lactoferrin is found in specific granules of neutrophils, which, following degranulation, are believed to be the main source of lactoferrin in blood plasma. It has been shown that lactoferrin concentration in the plasma increases during most inflammatory reactions and some viral infections (Kanyshkova et al. Biochemistry 2001, 66(1):1-7.).
Lactoferrin represents one of the first defense systems against pathogens, exhibiting antimicrobial, antibacterial, antiviral and antiparasitic activity. Lactoferrin was also found to influence immune system cells both positively and negatively. On one hand, it has been reported to support proliferation, differentiation, and activation of immune cells and strengthen the immune response. On the other hand, lactoferrin has also been reported to have anti-inflammatory properties, in reducing the production of some pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6), among others. In addition, lactoferrin has been reported to mediate inhibition of tumor growth and to possess several other biologic activities, including a ribonuclease activity (capable of RNA hydrolysis) and an osteogenic activity.
With the exception of iron binding, the biological activities of lactoferrin are thought to reside in a highly basic domain in the N-terminal region, designated lactoferricin. This part of the protein is released in the stomach at acidic pH by pepsin. Bovine lactoferricin is a highly potent 25aa peptide corresponding to residues 17-41 of lactoferrin, whereas the fragment released from human lactoferrin is larger (including positions 1-41) and has weaker antimicrobial properties. A number of lactoferricin derivatives have been described and tested, which retain at least a part of the activities of the native domain. An antimicrobial peptide derived from ovotransferrin, called OTAP-92, has also been identified, corresponding to positions 109-200 of ovotransferrin (Vogel et al., Biochem Cell Biol. 2002; 80(1):49-63).
Japanese Patent Application Publication No. JP 2004155751 discloses a peptide capable of suppressing the production of inflammatory cytokines such as TNF-α and IL-6, wherein the peptide used may be bovine lactoferrin hydrolyzed with a protease.
U.S. Patent Application Publication No. 2007/0197426 discloses polypeptide fragments of lactoferrin comprising the amino acid sequence of phenylalanine, lysine and aspartic acid, obtained by degradation with serine proteases such as elastase, wherein the molecular weight of said fragments is preferably less than 25 kDa. Specifically, disclosed are fragments of human lactoferrin of 21-25 kDa, with N-termini located at positions 240, 288 and 341. These fragments are described as having pro-inflammatory effects, such as inducing cytokine and chemokine production. The disclosed lactoferrin fragments are said to be distinct from pro-inflammatory lactoferrin fragments having a molecular weight of 30 to 60 kDa previously identified in bovine mastitis, reported by Japanese Patent Application Publication No. JP2003/289749. US '426 further discloses the production of four short synthetic peptides corresponding to positions 243-249, 251-259, 287-293 and 295-307 of human lactoferrin, wherein two of these peptides, namely those corresponding to positions 243-249 and 295-307, demonstrated an inflammation-promoting activity.
Komine et al. (Mol. Immunol. 2007 March; 44(7):1498-508) refers to certain lactoferrin fragments identified in parotid saliva of human periodontitis patients, with N-termini located at positions 4, 238, 286 and 340 for the 32, 23, 22 and 19 kDa fragments, respectively, characterized by low Con A affinity. The amounts of these fragments in saliva were reported to increase in periodontitis in association with the severity of the clinical symptoms. It is further indicated that a longitudal study is required to verify whether the elevated levels of these fragments subsequently decrease to the levels of healthy control subjects or whether persons who have high levels of these fragments may possess a predisposition to periodontal disease.
Komine et al. (J Vet Med. Sci. 2006 July; 68(7):715-23) identified fragments of 38, 23, 22 and 14 kDa, generated from elastase-treated bovine lactoferrin. The publication discloses that the amino acid sequences of the 22 and 23 kDa fragments correspond to positions 237-416 and 285-449 of bovine lactoferrin, respectively. These fragments were shown to have a pro-inflammatory activity, promoting cytotoxicity and leukocyte infiltration, through induction of pro-inflammatory cytokines and chemokines (TNF-α, IL-6, IL-8 and MCP-1), and suggested to take part in the initiation or progression of inflammation. The publication further discloses that measurements of the bovine lactoferrin concentration is used in Japan as a clinical marker for bovine mastitis in lactating cows.
Certain lactoferrin fragments having low Con A affinity and have also been described by Komine et al. (J Vet Med. Sci. 2006 March; 68(3):205-11; J Vet Med. Sci. 2005 July; 67(7):667-77) in healthy or mastitic mammary gland secretions. The N-terminal positions are 237, 285 and 240 for the 23, 22 and 19 kDa fragments, respectively. It is disclosed that the concentrations of these fragments increased in mastitis in correlation to the severity of the symptoms, and decreased after antibiotic therapy. In addition, considerable fluctuations in the concentration of the low Con A affinity fragments have been detected over time in drying cows.
Nowhere in the background art is it taught or disclosed that lactoferrin or fragments thereof may be used as molecular markers for defining the resolution phase of inflammation. There remains an unmet need for identification of molecular signals that differentiate between resolving and non-resolving inflammation, thereby facilitating a more suitable and effective treatment to inflammatory diseases.